Gyrase Cleavage Sites Research Articles

Bacterial Argonaute Proteins Aid Cell Division in the Presence of Topoisomerase Inhibitors in Escherichia coli.

Prokaryotic Argonaute (pAgo) proteins are guide-dependent nucleases that function in host defense against invaders. Recently, it was shown that TtAgo from Thermus thermophilus also participates in the completion of DNA replication by decatenating chromosomal DNA. Here, we show that two pAgos from cyanobacteria Synechococcus elongatus (SeAgo) and Limnothrix rosea (LrAgo) are active in heterologous Escherichia coli and aid cell division in the presence of the gyrase inhibitor ciprofloxacin, depending on the host double-strand break repair machinery. Both pAgos are preferentially loaded with small guide DNAs (smDNAs) derived from the sites of replication termination. Ciprofloxacin increases the amounts of smDNAs from the termination region and from the sites of genomic DNA cleavage by gyrase, suggesting that smDNA biogenesis depends on DNA replication and is stimulated by gyrase inhibition. Ciprofloxacin enhances asymmetry in the distribution of smDNAs around Chi sites, indicating that it induces double-strand breaks that serve as a source of smDNA during their processing by RecBCD. While active in E. coli, SeAgo does not protect its native host S. elongatus from ciprofloxacin. These results suggest that pAgo nucleases may help to complete replication of chromosomal DNA by promoting chromosome decatenation or participating in the processing of gyrase cleavage sites, and may switch their functional activities depending on the host species. IMPORTANCE Prokaryotic Argonautes (pAgos) are programmable nucleases with incompletely understood functions in vivo. In contrast to eukaryotic Argonautes, most studied pAgos recognize DNA targets. Recent studies suggested that pAgos can protect bacteria from invader DNA and counteract phage infection and may also have other functions including possible roles in DNA replication, repair, and gene regulation. Here, we have demonstrated that two cyanobacterial pAgos, SeAgo and LrAgo, can assist DNA replication and facilitate cell division in the presence of topoisomerase inhibitors in Escherichia coli. They are specifically loaded with small guide DNAs from the region of replication termination and protect the cells from the action of the gyrase inhibitor ciprofloxacin, suggesting that they help to complete DNA replication and/or repair gyrase-induced breaks. The results show that pAgo proteins may serve as a backup to topoisomerases under conditions unfavorable for DNA replication and may modulate the resistance of host bacterial strains to antibiotics.

Genome-wide mapping of fluoroquinolone-stabilized DNA gyrase cleavage sites displays drug specific effects that correlate with bacterial persistence.

Bacterial persisters are rare phenotypic variants that are suspected to be culprits of recurrent infections. Fluoroquinolones (FQs) are a class of antibiotics that facilitate bacterial killing by stabilizing bacterial type II topoisomerases when they are in a complex with cleaved DNA. In Escherichia coli, DNA gyrase is the primary FQ target, and previous work has demonstrated that persisters are not spared from FQ-induced DNA damage. Since DNA gyrase cleavage sites (GCSs) largely govern the sites of DNA damage from FQ treatment, we hypothesized that GCS characteristics (e.g. number, strength, location) may influence persistence. To test this hypothesis, we measured genome-wide GCS distributions after treatment with a panel of FQs in stationary-phase cultures. We found drug-specific effects on the GCS distribution and discovered a strong negative correlation between the genomic cleavage strength and FQ persister levels. Further experiments and analyses suggested that persistence was unlikely to be governed by cleavage to individual sites, but rather survival was a function of the genomic GCS distribution. Together, these findings demonstrate FQ-specific differences in GCS distribution that correlate with persister levels and suggest that FQs that better stabilize DNA gyrase in cleaved complexes with DNA will lead to lower levels of persistence.

Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome.

An important antibiotic target, DNA gyrase is an essential bacterial enzyme that introduces negative supercoils into DNA and relaxes positive supercoils accumulating in front of moving DNA and RNA polymerases. By altering the superhelical density, gyrase may regulate expression of bacterial genes. The information about how gyrase is distributed along genomic DNA and whether its distribution is affected by drugs is scarce. During catalysis, gyrase cleaves both DNA strands forming a covalently bound intermediate. By exploiting the ability of several topoisomerase poisons to stabilize this intermediate we developed a ChIP-Seq-based approach to locate, with single nucleotide resolution, DNA gyrase cleavage sites (GCSs) throughout the Escherichia coli genome. We identified an extended gyrase binding motif with phased 10-bp G/C content variation, indicating that bending ability of DNA contributes to gyrase binding. We also found that GCSs are enriched in extended regions located downstream of highly transcribed operons. Transcription inhibition leads to redistribution of gyrase suggesting that the enrichment is functionally significant. Our method can be applied for precise mapping of prokaryotic and eukaryotic type II topoisomerases cleavage sites in a variety of organisms and paves the way for future studies of various topoisomerase inhibitors.

Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome.

An important antibiotic target, DNA gyrase is an essential bacterial enzyme that introduces negative supercoils into DNA and relaxes positive supercoils accumulating in front of moving DNA and RNA polymerases. By altering the superhelical density, gyrase may regulate expression of bacterial genes. The information about how gyrase is distributed along genomic DNA and whether its distribution is affected by drugs is scarce. During catalysis, gyrase cleaves both DNA strands forming a covalently bound intermediate. By exploiting the ability of several topoisomerase poisons to stabilize this intermediate we developed a ChIP-Seq-based approach to locate, with single nucleotide resolution, DNA gyrase cleavage sites (GCSs) throughout the Escherichia coli genome. We identified an extended gyrase binding motif with phased 10-bp G/C content variation, indicating that bending ability of DNA contributes to gyrase binding. We also found that GCSs are enriched in extended regions located downstream of highly transcribed operons. Transcription inhibition leads to redistribution of gyrase suggesting that the enrichment is functionally significant. Our method can be applied for precise mapping of prokaryotic and eukaryotic type II topoisomerases cleavage sites in a variety of organisms and paves the way for future studies of various topoisomerase inhibitors.

Topoisomerase Action on Short DNA Duplexes Reveals Requirements for Gate and Transfer DNA Segments

Type II topoisomerases change DNA topology by passage of one DNA duplex (the transfer, T-segment) through a transient double-stranded break in another (the gate, G-segment). Here we monitor the passage between short double-stranded DNA segments within long single-stranded DNA circles that leads to catenation of the circles. To facilitate catenation, the circles were brought into close proximity using a tethering oligonucleotide, which was removed after the reaction was complete. We varied the length and the composition of the reacting DNA segments. The minimal DNA duplex length at which we detected catenation was 50-60 bp for DNA gyrase and 40 bp for topoisomerase IV (Topo IV). For Topo IV, catenation was observed when one, but not both, of the DNA-DNA duplexes was replaced by a DNA-RNA duplex. Topo IV cleaved the DNA-DNA duplex, but not the DNA-RNA duplex implying that the DNA-RNA duplex can be a T-segment but not a G-segment.

DNA gyrase can cleave short DNA fragments in the presence of quinolone drugs.

We have analysed the DNA cleavage reaction of DNA gyrase using oligonucleotides annealed to a single-stranded M13 derivative containing a preferred gyrase cleavage site. We find that gyrase can cleave duplexes down to approximately 20 bp in size in the presence of the quinolone drugs ciprofloxacin and oxolinic acid. Ciprofloxacin shows a variation in its site specificity with an apparent preference for G bases adjacent to the cleavage sites, whereas oxolinic acid stimulates cleavage predominantly at the previously determined site. With either drug, cleavage will not occur within 6 bases from the end of a DNA duplex or a nick. We suggest that cleavage site specificity with short DNA duplexes is determined by drug-DNA interactions whereas with longer fragments the positioning effect of the DNA wrap around gyrase prescribes the site of cleavage.

Effect of netropsin, distamycin A and chromomycin A 3 on the binding and cleavage reaction of DNA gyrase

The influence of netropsin (Nt), distamycin A (Dst-3) and chromomycin A 3 (CHR) on the binding of gyrase from Streptomyces noursei to an 162 bp-fragment of pBR 322 containing a strong gyrase cleavage site and on the gyrase mediated cleavage of this fragment was analyzed. Binding of the enzyme to the fragment is effectively inhibited by the GC-specific drug CHR, but poorly influenced by Dst-3, while Nt is ineffective. Cleavage of the fragment catalysed by the enzyme is inhibited by all three ligands but to different extent. Dst-3 and Nt inhibit the enzyme cleavage reaction at 20- or 250-fold higher concentration than that required for CHR. The inhibitory mechanism of CHR on gyrase-DNA binding and cleavage may be related to a competitive interaction of the ligand to GC sequences located at and around the gyrase cleavage site. The fact that AT-specific minor groove binders Dst-3 and Nt poorly inhibit the binding of gyrase to the fragment due to the low amount of the AT basepair sequences contained in the fragment and their inhibitory influence on the cleavage step underlines the role of the DNA minor groove during enzyme action.

The Complex of DNA Gyrase and Quinolone Drugs with DNA Forms a Barrier to Transcription by RNA Polymerase

The effects of DNA gyrase and quinolone drugs on in vitro transcription of a template containing a preferred gyrase cleavage site have been investigated. We have found that gyrase-quinolone complexes with DNA lead to blocking of transcription by Escherichia coli and bacteriophage T7 RNA polymerases. Either gyrase or quinolone alone has no effect on transcription. With DNA gyrase containing a point mutation in the gyrase A protein, known to confer quinolone resistance, blocking was found to occur only at much higher concentrations of the drug. Other agents that inhibit gyrase-catalysed supercoiling (novobiocin and 5′-adenylyl-β, γ-imidodiphosphate) do not arrest transcription in the presence of gyrase. Mapping of the transcription termination sites in the presence of gyrase and quinolones shows that blocking occurs about 10 to 20 base-pairs upstream of the gyrase cleavage site. Analysis of transcription in the absence of drug suggests that RNA polymerase does not displace gyrase from the template. These results are discussed in the light of models for the bactericidal effects of quinolone drugs.

Evidence for a conformational change in the DNA gyrase-DNA complex from hydroxyl radical footprinting.

We have used the technique of hydroxyl radical footprinting to probe the complex between DNA gyrase and a 198 bp DNA fragment containing the preferred gyrase cleavage site from plasmid pBR322. We find that gyrase protects 128 bp from the hydroxyl radical with the central 13 bp (adjacent to the gyrase cleavage site) being most strongly protected. Flanking the central region are arms showing periodic protection from the reagent suggesting a helical repeat of 10.6 bp, consistent with the DNA being wrapped upon the enzyme surface. The presence of 5'-adenylyl-beta,gamma-imidodiphosphate or a quinolone drug causes alteration of the protection pattern consistent with a conformational change in the complex involving one arm of the wrapped DNA. The significance of these results for the mechanism of DNA supercoiling by gyrase is discussed.

The cleavage of DNA at phosphorothioate internucleotidic linkages by DNA gyrase.

We have constructed a plasmid which contains 22 copies of a 147 bp DNA fragment which contains the major DNA gyrase cleavage site from plasmid pBR322 (located at base-pair 990). We have found that this fragment is efficiently bound and cleaved by gyrase. The selectivity for the sequence corresponding to position 990 in pBR322 is maintained even when this site is located only 15 bp from one end of the 147 bp fragment. A strategy for the specific incorporation of a single thiophosphoryl linkage into the 147 bp fragment has been developed, and gyrase has been shown to catalyse efficient cleavage of fragments bearing phosphorothioate linkages at the gyrase cleavage site in one or both strands.

Sequence specificity of Bacillus subtilis DNA gyrase in vivo.

Linearization of pBG0 (a hydrid between Escherichia coli plasmid pBR322 and Staphylococcus aureus plasmid pUB110) was performed by lysis of the oxolinic acid treated Bacillus subtilis protoplasts with sodium dodecyl sulfate. This plasmid DNA linearization was used both for a detailed mapping of DNA gyrase cleavage sites of various strength and for the nucleotide sequence determinations at the points of gyrase-mediated scissions by introducing the XhoI linker DNA. A total of 40 plasmids carrying inserted XhoI linker were sequenced by labeling 3' termini of XhoI sites; 38 of them were found to contain a duplication of four base-pairs of the plasmid sequence flanking the linker, which were characteristic of the oxolinic acid-induced DNA cleavage by E. coli DNA gyrase in vitro and in vivo. The relative strength of these sequenced sites was established by comparing their positions to the sites mapped on the appropriate plasmid genome. This allowed us to propose a consensus sequence of B. subtilis DNA gyrase in vivo cleavage site: [sequence: see text] where N is any nucleotide. The bases in parentheses were preferred secondarily. The involvement of DNA gyrase in illegitimate recombination events in Bacillus subtilis is discussed.

Effects of transcription and translation on gyrase-mediated DNA cleavage in Escherichia coli.

Gyrase-mediated DNA cleavage on plasmid DNAs was measured in Escherichia coli treated with oxolinic acid. On pBR322 DNA, gyrase cleavage sites were concentrated in the region between the 3'-ends of the tetA and bla genes. The preferential cleavage in this region was dependent on RNA transcription and the divergent orientation of these two transcription units. The enhanced gyrase cleavage also required translation; chloramphenicol treatment or the insertion of a translation terminator within the 5'-proximal region of the tetA gene abolished the enhanced cleavage. We suggest that the enhanced gyrase cleavage may reflect the changes in local DNA supercoiling during RNA transcription as gyrase cleavage in vitro was shown to be sensitive to the supercoiling state of DNA. The effects of transcription and translation on gyrase cleavage can best be explained by the twin-supercoiled-domain model of transcription (Liu, L. F., and Wang, J. C. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7024-7027).

DNA gyrase binds to the family of prokaryotic repetitive extragenic palindromic sequences.

A family of repetitive extragenic palindromic (REP) sequences is composed of hundreds of copies distributed throughout the chromosome. Their palindromic nature and conservation suggested that they are specifically recognized by a protein(s). We have identified DNA gyrase [DNA topoisomerase (ATP-hydrolysing), EC 5.99.1.3] as one of the REP-binding proteins. Gyrase has at least a 10-fold higher affinity for DNA containing REP sequences than for DNA not containing REP sequences. Binding effectiveness correlates directly with the number of REP sequences in the DNA. DNase I footprinting shows that gyrase protects 205 base pairs on a REP-containing DNA fragment enclosing the REP sequences. In agreement with the above results, a comparison of the REP consensus sequence with the sequence of previously identified pBR322 "strong" gyrase cleavage sites reveals a high degree of homology. Because REP sequences are numerous and found throughout the genome, we suggest they have physiological functions mediated through their interaction with gyrase, such as being sites of action for the maintenance of DNA supercoiling. In addition, we speculate that these interactions may be of a structural nature, such as involvement in the higher-order structure of the bacterial chromosome.

DNA gyrase complex with DNA: determinants for site-specific DNA breakage.

DNA gyrase catalyses DNA supercoiling by making a transient double-stranded DNA break within its 120-150 bp binding site on DNA. Addition of the inhibitor oxolinic acid to the reaction followed by detergent traps a covalent enzyme-DNA intermediate inducing sequence-specific DNA cleavage and revealing potential sites of gyrase action on DNA. We have used site-directed mutagenesis to examine the interaction of Escherichia coli gyrase with its major cleavage site in plasmid pBR322. Point mutations have been identified within a short region encompassing the site of DNA scission that reduce or abolish gyrase cleavage in vitro. Mapping of gyrase cleavage sites in vivo reveals that the pBR322 site has the same structure as seen in vitro and is similarly sensitive to specific point changes. The mutagenesis results demonstrate conclusively that a major determinant for gyrase cleavage resides at the break site itself and agree broadly with consensus sequence studies. The gyrase cleavage sequence alone is not a good substrate, however, and requires one or other arm of flanking DNA for efficient DNA breakage. These results are discussed in relation to the mechanism and structure of the gyrase complex.

Mapping of DNA gyrase cleavage sites in vivo oxolinic acid induced cleavages in plasmid pBR322

We have developed a procedure which permits the mapping of DNA gyrase cleavage sites in vivo. Addition of oxolinic acid, an inhibitor of DNA gyrase, to growing cells of Escherichia coli containing the plasmid pBR322 resulted in double-strand cleavage of DNA, and allowed the isolation of significant quantities of linearized plasmid DNA after lysis of treated cells with sodium dodecyl sulfate. Initially the linear product was purified from agarose gels, cleaved by restriction endonucleases, and then subjected to Southern hybridization analysis using defined DNA probes. A number of distinct cleavage sites, used with varying degrees of efficiency, were identified within pBR322 using this simple procedure. To achieve greater resolution and to improve sensitivity, we then employed an electroblotting procedure to transfer DNA fragments from acrylamide gels onto nylon membranes. This alternative method does not require the isolation of the linearized product before performing the mapping procedure. The improved resolution obtained from acrylamide gels and the superior binding properties of the nylon membranes have allowed us to accurately map 74 distinct oxolinic acid-induced cleavage sites within pBR322. The significance of these findings in light of previously reported studies in vitro, as well as the possible role of such sites during illegitimate recombination, are discussed.

Sites of reaction of Escherichia coli DNA gyrase on pBR322 in vivo as revealed by oxolinic acid-induced plasmid linearization

pBR322 DNA, linearized by lysis of an oxolinic acid-treated culture of Escherichia coli strain DK6recA − (pBR322) with sodium dodecyl sulfate, was purified, treated with DNA polymerase in the presence of the four deoxynucleoside triphosphates, and ligated to DNA linkers containing the XhoI recognition sequence. Most of the drug-resistant colonies resulting from transformation of E. coli with this material bore plasmids that appeared by restriction enzyme analysis to differ from pBR322 only by the introduction of an XhoI site. The XhoI sites in plasmids from 93 transformants were distributed unevenly around the pBR322 map. Maxam-Gilbert DNA sequence analysis of 36 of these plasmids, labeled at the 5′ termini of the XhoI sites, revealed that 29 of them contained, in addition to the XhoI linker, a duplication of four base-pairs of the pBR322 sequence surrounding the linker. Therefore, oxolinic acid-induced linearization must have resulted in 5′-terminal extensions of four bases, the configuration known to result from oxolinic acid-induced DNA cleavage by DNA gyrase in vitro. The sequence data thus allowed the determination of the precise point at which linearization occurred, apparently by the abortion of a gyrase-DNA covalent intermediate that existed in vivo. When the 19 different sites of the 29 plasmids were compared, the following set of rules could be derived: ▪ where N is any nucleotide, R is a purine, and Y is a pyrimidine. Cleavage occurred at the line between the eighth and ninth positions from the left. The parenthetical G and T were preferred secondarily to T and G, respectively, whereas T and G in the 13th position from the left were equally preferred. Several of these rules are similar to those proposed previously based on several in vitro gyrase cleavage sites. Some of our rules show dyad symmetry around the axis midway between the cleavage points in the two strands, while others are distinctly asymmetric.

Mechanism of illegitimate recombination: common sites for recombination and cleavage mediated by E. coli DNA gyrase.

Illegitimate recombination dependent on DNA gyrase in a cell-free system has previously been described. We have now mapped DNA gyrase cleavage sites in the vicinity of known recombination sites in pBR322. Among five recombination sites examined, three were found to coincide with a DNA gyrase cleavage site. This result suggests that the cleavage of DNA by DNA gyrase has a central role in the recombination process.